Confocal Microscope Advances Cadet Cardiovascular Research

Researchers at VMI are working towards uncovering the secrets of blood vessel development and maintenance using a state-of-the-art confocal microscope located at Washington and Lee University.

Cadet Fritz Dawson ’13 is examining the role that estrogen plays in blood vessel development and, in a related project, Cadet Pete VanSteyn ’13 is looking into the importance of nitric oxide in blood vessel development. Both cadets are using developing zebrafish, which are prime candidates for study for many reasons, including the fact that they have relatively simple and regular blood vessel patterns.

“‘Why would anyone be working with a fish,’ some ask, ‘when you’re interested in the human heart and blood vessels?’” said Col. Jim Turner ’65, head of VMI’s biology department and mentor for the cadet research projects. Turner explained that what is learned from studying zebrafish blood vessels should be applicable to human blood vessels.

“The belief is that estrogen plays a key role in micro concentrations in both blood vessel development and blood vessel maintenance,” said Dawson. “We use the zebrafish for a few reasons. They grow really fast, they’re transparent so we can see their blood vessels and internal anatomy, and they can internalize chemical solutions through their skin very quickly.”

Researchers at VMI are working with a type of zebrafish that has been genetically altered to optimize their usefulness in blood vessel research.

“They have a jellyfish protein gene inserted into their DNA, so that makes their developing blood vessels glow bright green when they’re hit with a certain wavelength of light,” said Dawson. “When you look at them normally they just look like a normal fish, but when they’re hit with the laser, all of a sudden you see all of their blood vessels light up.”

Dawson worked with three groups of zebrafish: a control group, a group that received an estrogen supplement, and a group that was treated with an aromatase inhibitor, which made the fish unable to produce estrogen.

“What we found is that when estrogen was restricted, the blood vessels would all deteriorate. You can see a significant difference in the normal blood vessel structure. It looks like someone went in there with a weed whacker and cut all these gaps,” said Dawson. “The fish that received the estrogen supplement showed increased angiogenesis, or blood vessel growth.”

Just noticing that the blood vessels were deteriorating wasn’t enough; Dawson had to find a way to precisely quantify the levels of deterioration to produce a usable set of data.

“With his creativity and imagination, he was the first person to find a way to get some quantitative data on the development of blood vessels,” said Turner. “He does that so precisely that his data has already shown a significant difference in the treatment types.”

Among the factors that Dawson considers are numbers of gaps and the thickness of blood vessels. To do that he needs an image that shows all the blood vessels isolated and in focus. That’s what the confocal microscope makes possible.

“We have the technology here at VMI to view the blood vessels in real time, but what the equipment at W&L allows us to do is take a z-stack, which is a stack of images taken of different focal points to produce a composite image,” said Dawson. “So by doing that you can see all the blood vessels at once and quantify the level of blood vessel development.”

Dawson is currently working on an Institute Honors thesis on his research, and a manuscript has been accepted by the refereed journal General and Comparative Endocrinology. Dawson was one of 10 undergraduates contributing to the paper, “Estrogen Prevents Cardiac and Vascular Failure in the ‘Listless’ Zebrafish Developmental Model.”

“Pete VanSteyn wanted to dig deeper into the process that’s going on here,” said Turner. “We know that estrogen has a direct effect on the synthesis of a unique molecule called nitric oxide.

“Nitric oxide has powerful influences on the cardiovascular system,” said Turner. “VanSteyn’s results this summer indicated that you can treat the fish with nitric oxide inhibitors and get the same or even more pronounced results than Fritz saw with the estrogen inhibitors, and if you add estrogen back, then you get recovery.”

This line of research is likely to be picked up by cadet researchers for years to come.

“We’re trolling down the possible pathway for an actual mechanism, and we think we’re getting very close with the nitric oxide paradigm,” said Turner. “Then we can go further into the cell and see what nitric oxide is actually doing.”

The research is another example of VMI cadets making real contributions to scientific discourse.

“No one can tell you that undergraduates can’t do high-end science work and can’t be trusted with equipment worth hundreds of thousands of dollars,” said Turner. “They can, and they do. I’ve had the pleasure and the honor to include all the undergraduates that I work with on my scientific publications as co-authors because they all add important bits and pieces to the overall project.”

That high-end science work is possible, in part, because of the equipment available to cadets. “The quality of the equipment we have here is just as good as what you would find in a research lab at a large university,” said Turner. “Our philosophy is that we have this nice equipment and we’re certainly not going to keep the cadets from it.”

The confocal microscope was acquired last year through a $366,000 National Science Foundation grant. VMI professors were among those who contributed project descriptions to the proposal for that grant, according to W&L.

“We have this wonderful scope that was set up last year at Washington and Lee due to the collaboration of faculty members at both institutions,” said Turner. “You can imagine how convenient it is for cadets who have a restricted lifestyle just to walk a block or so to use this piece of equipment.”

Prior to the acquisition of the confocal microscope housed at Washington and Lee, students had to travel to James Madison University to use a confocal microscope, which made research during the semester difficult.

The VMI experience is framed by relevant education within a military environment. It’s an education that develops character, fosters a lifelong appreciation of physical fitness,engenders a sense of responsibility to others, and cultivates
the ability to master adversity.